This invention relates generally to the field of recombinant genetics, and specifically to the use of a meiosis-specific promoter for regulating the expression of genes in plants. More particularly, the invention enables the expression of desired introduced genes in transgenic plants to be controlled in a developmentally related manner.
To optimize crop production, the agricultural industry relies on plants having certain desirable traits relating to growth characteristics, resistance to disease and pest infestation, and yield.
One way to attain this goal is to produce hybrid plants possessing the desirable traits of parental plants. To insure the uniformity of hybrid seed, pollination control methods must be employed which favor cross-pollination and eliminate self-pollination. Such methods of inhibiting self-pollination of plants typically include mechanical means, as well as chemical.
Mechanical means such as detasseling, the process currently used for plants such as corn, is labor intensive and costly to perform. For some plants, for example those whose male and female gametophytes are within the same flower, the method is even less desirable.
As an alternative to mechanical methods to prevent self pollination, chemicals which kill or inhibit formation of functional pollen may be used. Application of chemicals, however, may result in overall toxicity to the plant or to the environment. Another limitation of the chemical means is the possibility that one application will not be effective for plants which have an extended period of flower formation.
More recently, the industry has relied on pollen-control-based systems which involve genetic manipulation of the events involved in the development of functional pollen. One example is described in U.S. Pat. No. 5,689,051, the contents of which are hereby incorporated by reference in their entirety. Briefly, regulatory elements are described which alter the expression of genes involved in pollen development. The ability to interfere with the production or formation of pollen results in a plant that is male sterile and therefore, incapable of self-pollination.
Similarly, it is possible to control plant characteristics other than pollen development by manipulating the control of gene expression which gives rise to the desired phenotype. An example of this technology is illustrated in U.S. Pat. No. 5,723,765. It discloses a system in which the expression of genes can be controlled by external stimuli or in a developmentally related fashion. The system entails creating a transgenic plant containing a series of functionally interrelated DNA sequences, including a promoter that is active at a particular stage in plant development or in response to an external stimulus, for example, a particular environmental condition. Hence, the need for sequence information for developmental stage-specific promoters is evident.
Male reproductive development and ultimately, pollen formation in plants begins with meiosis. Meiosis consists of a complex set of processes involving homologous chromosome pairing, synapsis, chiasma formation and crossing over, chromosome segregation, and reductional division. In male meiosis of flowering plants, the product of meiosis is a callose-enclosed tetrad of haploid microspores, the precursors of pollen grains. Manipulation of gene expression using a genetic regulatory element such as the one described herein, whose role occurs early on in pollen development, has significant implications.
From the many meiotic mutants described in plants and other organisms, it is obvious that meiosis is an extremely complex event involving a large number of genes. While the dissection of meiosis by the isolation and characterization of mutants and the genes responsible has progressed in yeast and Drosophila, at present little is known of the molecular details of male meiosis and the very early development of microspores in flowering plants. Recently, however, several mutants have been described in Arabidopsis thaliana in which male gametophyte development is affected. Specifically, a T-DNA induced male-sterile mutant of Arabidopsis thaliana that is defective in meiosis and that produces tetrads that are abnormal in number and in size has been described by He et al., Sex. Plant Reproduction 9: 54-57 (1996). In this mutant, instead of the normal tetrad of four microspores being formed after meiosis, a xe2x80x9ctetradxe2x80x9d consisting of from 5 to 8 microspores is formed. The microspores show a wide range of sizes and of DNA contents. Plants homozygous for the mutation were reported to be male sterile and not to produce filled siliques, although occasionally a few siliques containing a few seeds are produced by some homozygous mutant plants.
T-DNA insertional mutagenesis can be used to produce mutant plants. An Agrobacterium tumefaciens strain containing the cointegrate pGV3850: 1003 Ti plasmid with NOS and NPT plant markers and a bacterial NPT marker was used to create the Arabidopsis mutants described above. The disruption of the plant""s normal genomic DNA by the insertion of a T-DNA gives rise to a non-functional gene and mutant phenotype. One of the resulting phenotypes is plants that are male-sterile, a desirable trait for the development of hybrid seed.
A common feature of Agrobacterium-mediated plant transformation is the appearance of T-DNA duplications and rearrangements in the plant genome. In other words, the insertion of the T-DNA vector is random and does not produce consistent and predictable results. To reproduce by mutagenesis the desired mutation in other plants would not be possible without undue experimentation. Information about a gene sequence involved in the meiotic events giving rise to viable pollen provides a new mechanism for manipulating those events and producing plants which are male sterile.
The current invention relates to a gene, MEI1, and its promoter that are involved in meiosis. Significantly, the gene seems only to function at male meiosis. A mutation in the MEI1 gene results in a plant that is male sterile, but female fertile. A mutant line of Arabidopsis, designated 6492, has a phenotype similar to plants carrying the MEI1 sequence, but is female sterile, indicating the involvement of a gene different from the MEI1 sequence described herein. The MEI1 gene is novel in that, even though there is a sequence of over 100 base pairs available beyond the 3xe2x80x2 stop codon TAA, there is no evidence of a 3xe2x80x2 end poly(A) tail of this gene. Because this gene has unique splicing signals, it may not be polyadenylated the same way that normal genes usually are.
Knowledge of the MEI1 gene sequence and its promoter provides new ways of producing transgenic plants possessing desired characteristics. The promoter for the MEI1 gene can be used to facilitate the expression of exogenous genes, for example, suicide genes, in a manner related directly to pollen development. The MEI1 gene itself can be used to facilitate the isolation of homologous genes in other plant species. An artificial gene coding for an antisense RNA can be introduced to prevent expression of the specific target gene to whose mRNA it is complementary, in this case, the MEI1 gene product crucial for meiosis.
The invention relates to an isolated DNA sequence comprising a nucleotide sequence capable of regulating the expression of a second DNA sequence when the DNAs are part of a recombinant DNA construct. The isolated molecule includes the nucleotide sequence of the promoter region derived from a meiosis specific gene, MEI1, of Arabidopsis thaliana having the sequence (SEQ ID NO.1) described in FIG. 1.
In another aspect, the invention relates to a vector comprising the isolated DNA molecule and further comprising a DNA sequence encoding a gene product. The DNA sequence is operably linked to the promoter.
In yet another aspect, the invention relates to the expression of a gene product which disrupts the function or formation of pollen. The DNA sequence to be expressed may be heterologous with respect to the promoter.
In still another aspect, the invention relates to a transgenic plant comprising the isolated DNA promoter sequence of the invention.
In yet another related aspect, the invention provides for the production of a transformed plant cell comprising the isolated DNA sequence and subsequently the growth of transgenic plants containing the characteristic phenotype bestowed by the expression of the gene inserted into the genome of the transformed cell.
In another aspect, the invention relates to a method of producing a male sterile plant, comprising the steps of: (1) providing a transformed plant that contains a recombinant DNA molecule that includes (a) a DNA sequence which inhibits pollen formation or function by altering meiosis; and (b) a MEI1 or homologous promoter from other plants that is operatively linked to the DNA sequence of the invention; and (2) growing the plant under conditions such that male sterility is achieved as a result of the recombinant DNA sequence.
In yet another aspect, the invention relates to a plant containing a recombinant DNA molecule which includes at least two elements: (1) a DNA sequence that inhibits pollen formation or function by altering meiosis; and (2) the MEI1 promoter. The MEI1 promoter is operatively linked to the DNA sequence that inhibits pollen formation or function by altering meiosis.